Project Summary/Abstract Inositol 1,4,5-trisphosphate receptors (IP3Rs) are the primary intracellular Ca2+ release channels in non- excitable cells. IP3R activity drives numerous cellular processes including cell division, migration, mitochondrial bioenergetics, autophagy, apoptosis and necrosis. Due to its diverse roles in cell fate decisions, it is perhaps unsurprising that tumor cells can gain a selective advantage by co-opting and modifying the IP3R-mediated Ca2+ signaling networks that underlie these processes. One mechanism that cells use to escape programmed cell death is to increase expression of anti-apoptotic BCL2 proteins, including Bcl-2, Bcl-xL, and Mcl-1. Among their various anti-apoptotic roles in cells, BCL2 proteins bind to IP3R and modulate Ca2+ dynamics. Despite extensive characterization of the role of IP3R in apoptosis and the anti-apoptotic effects of BCL2-IP3R interactions, mechanistic understanding of IP3R gating and modulation by BCL2 proteins is poorly understood at the molecular level. It is clear, however, that BCL2-IP3R interactions promote cell survival in response to apoptotic insult. Interactions between BCL2 and IP3R throttle the cell between enhanced mitochondrial bioenergetics through activation of Ca2+-dependent enzymes and metabolites, and cell death through large Ca2+ flux into the mitochondrial, leading to mitochondrial Ca2+ overload and outer membrane permeabilization, the commitment step in apoptosis. In the proposed work, functional and structural approaches will be employed to establish a mechanistic understanding of activation of IP3R by Ca2+ and IP3, and inhibition of IP3R by high Ca2+ concentrations. Furthermore, through single channel studies and solution of a BCL2-IP3R complex structure, a mechanistic basis for the regulation of IP3R by BCL2 proteins will be developed. The work will provide a new framework by which we can understand IP3R-mediated cell death, paving the way for new understanding and the design of therapeutics.